Caffeic Acid Phenethyl Ester and Caffeamide Derivatives Suppress Oral Squamous Cell Carcinoma Cells

Caffeic acid phenethyl ester (CAPE) contains antibiotic and anticancer activities. Therefore, we aimed to investigate the anticancer properties and mechanisms of CAPE and caffeamide derivatives in the oral squamous cell carcinoma cell (OSCC) lines SAS and OECM-1. The anti-OSCC effects of CAPE and the caffeamide derivatives (26G, 36C, 36H, 36K, and 36M) were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. Cell cycle and total reactive oxygen species (ROS) production were analyzed using flow cytometry. The relative protein expression of malignant phenotypes was determined via Western blot analysis. The results showed that 26G and 36M were more cytotoxic than the other compounds in SAS cells. After 26G or 36M treatment for 48 h, cell cycle S phase or G2/M phase arrest was induced, and cellular ROS increased at 24 h, and then decreased at 48 h in both cell lines. The expression levels of cell cycle regulatory and anti-ROS proteins were downregulated. In addition, 26G or 36M treatment inhibited malignant phenotypes through mTOR-ULK1-P62-LC3 autophagic signaling activated by ROS generation. These results showed that 26G and 36M induce cancer cell death by activating autophagy signaling, which is correlated with altered cellular oxidative stress.


Introduction
Oral cancer is a common type of head and neck cancer, with an estimated 650,000 new cases reported annually worldwide. Oral squamous cell carcinoma (OSCC) is a malignant tumor originating from squamous cells lining the oral cavity, such as the lips, hard palate, gums, tongue, and cheeks. The prevalence of OSCC varies with geographic location, with higher rates observed in some areas of Asia, South America, and Africa. A previous study indicated that oral cancer is highly prevalent in Asia-Pacific men, owing to the use of

The Cytotoxicity of 26G and 36M in Oral Cancer Cell Lines
First, the cytotoxicity of the CAPE and 5 caffeamide derivatives (26G, 36C, 36H, 36K, and 36M) was tested in the SAS cell line. The cells were treated with the vehicle or 12.5-200 µM of each compound for 24 h to determine the half-maximal inhibitory concentration (IC50). The chemical structures, molecular weights, and IC50 values of the SAS cell lines are presented in Table 1. The IC50 values of 36C, 36K, and 36H were greater than 200 µM. SAS cells were more sensitive to 26G and 36M than to the other compounds. Therefore, this study focused on analyzing the anticancer potentials of 26G and 36M. SAS cells were more sensitive to 26G and 36M than to the other compounds. Therefore, this study focused on analyzing the anticancer potentials of 26G and 36M. The dose-and time-dependent cytotoxicities of 26G and 36M were analyzed in SAS and OECM-1 cell lines. Both SAS and OECM-1 cells showed a decreased cell viability rate upon treatment with 26G and 36M at 25, 50, or 100 μM for 24, 48, and 72 h, compared to that of the vehicle control group (Figure 1). The cytotoxicity of 26G and 36M were doseand time-dependent increased in the SAS cell line ( Figure 1A) and OECM-1 ( Figure 1B). The inhibitory effect on cancer cells showed that 36M was better than 26G for 48 and 72 h, although the IC50 of 26G was lower than the IC50 of 36M (Table 1). Based on the cell viability rate at the same dose, we found that 36M exhibited more powerful anticancer ability than 26G did by inducing cell toxicity. SAS cells were more sensitive to 26G treatment for 24 h than OECM-1 cells were. However, the cytotoxicity of 26G was stronger for OECM-1 at 48 and 72 h.  Table 1. The IC50 values of 36C, 36K, and 36H were greater than 200 μM. SAS cells were more sensitive to 26G and 36M than to the other compounds. Therefore, this study focused on analyzing the anticancer potentials of 26G and 36M. The dose-and time-dependent cytotoxicities of 26G and 36M were analyzed in SAS and OECM-1 cell lines. Both SAS and OECM-1 cells showed a decreased cell viability rate upon treatment with 26G and 36M at 25, 50, or 100 μM for 24, 48, and 72 h, compared to that of the vehicle control group (Figure 1). The cytotoxicity of 26G and 36M were doseand time-dependent increased in the SAS cell line ( Figure 1A) and OECM-1 ( Figure 1B). The inhibitory effect on cancer cells showed that 36M was better than 26G for 48 and 72 h, although the IC50 of 26G was lower than the IC50 of 36M (Table 1). Based on the cell viability rate at the same dose, we found that 36M exhibited more powerful anticancer ability than 26G did by inducing cell toxicity. SAS cells were more sensitive to 26G treatment for 24 h than OECM-1 cells were. However, the cytotoxicity of 26G was stronger for OECM-1 at 48 and 72 h.  The dose-and time-dependent cytotoxicities of 26G and 36M were analyzed in SAS and OECM-1 cell lines. Both SAS and OECM-1 cells showed a decreased cell viability rate upon treatment with 26G and 36M at 25, 50, or 100 μM for 24, 48, and 72 h, compared to that of the vehicle control group (Figure 1). The cytotoxicity of 26G and 36M were doseand time-dependent increased in the SAS cell line ( Figure 1A) and OECM-1 ( Figure 1B). The inhibitory effect on cancer cells showed that 36M was better than 26G for 48 and 72 h, although the IC50 of 26G was lower than the IC50 of 36M (Table 1). Based on the cell viability rate at the same dose, we found that 36M exhibited more powerful anticancer ability than 26G did by inducing cell toxicity. SAS cells were more sensitive to 26G treatment for 24 h than OECM-1 cells were. However, the cytotoxicity of 26G was stronger for OECM-1 at 48 and 72 h. The dose-and time-dependent cytotoxicities of 26G and 36M were analyzed in SAS and OECM-1 cell lines. Both SAS and OECM-1 cells showed a decreased cell viability rate upon treatment with 26G and 36M at 25, 50, or 100 μM for 24, 48, and 72 h, compared to that of the vehicle control group (Figure 1). The cytotoxicity of 26G and 36M were doseand time-dependent increased in the SAS cell line ( Figure 1A) and OECM-1 ( Figure 1B). The inhibitory effect on cancer cells showed that 36M was better than 26G for 48 and 72 h, although the IC50 of 26G was lower than the IC50 of 36M (Table 1). Based on the cell viability rate at the same dose, we found that 36M exhibited more powerful anticancer ability than 26G did by inducing cell toxicity. SAS cells were more sensitive to 26G treatment for 24 h than OECM-1 cells were. However, the cytotoxicity of 26G was stronger for OECM-1 at 48 and 72 h.  The dose-and time-dependent cytotoxicities of 26G and 36M were analyzed in SAS and OECM-1 cell lines. Both SAS and OECM-1 cells showed a decreased cell viability rate upon treatment with 26G and 36M at 25, 50, or 100 μM for 24, 48, and 72 h, compared to that of the vehicle control group (Figure 1). The cytotoxicity of 26G and 36M were doseand time-dependent increased in the SAS cell line ( Figure 1A) and OECM-1 ( Figure 1B). The inhibitory effect on cancer cells showed that 36M was better than 26G for 48 and 72 h, although the IC50 of 26G was lower than the IC50 of 36M (Table 1). Based on the cell viability rate at the same dose, we found that 36M exhibited more powerful anticancer ability than 26G did by inducing cell toxicity. SAS cells were more sensitive to 26G treatment for 24 h than OECM-1 cells were. However, the cytotoxicity of 26G was stronger for OECM-1 at 48 and 72 h. The dose-and time-dependent cytotoxicities of 26G and 36M were analyzed in SAS and OECM-1 cell lines. Both SAS and OECM-1 cells showed a decreased cell viability rate upon treatment with 26G and 36M at 25, 50, or 100 µM for 24, 48, and 72 h, compared to that of the vehicle control group (Figure 1). The cytotoxicity of 26G and 36M were doseand time-dependent increased in the SAS cell line ( Figure 1A) and OECM-1 ( Figure 1B). The inhibitory effect on cancer cells showed that 36M was better than 26G for 48 and 72 h, although the IC50 of 26G was lower than the IC50 of 36M (Table 1). Based on the cell viability rate at the same dose, we found that 36M exhibited more powerful anticancer ability than 26G did by inducing cell toxicity. SAS cells were more sensitive to 26G treatment for 24 h than OECM-1 cells were. However, the cytotoxicity of 26G was stronger for OECM-1 at 48 and 72 h.

The 26G and 36M Treatments Increased G1 Phase and S Phase Arrest
Analyzing the effects of 26G and 36M on the cell cycle is important for understanding their anticancer mechanisms. PI staining and flow cytometry were performed to analyze whether 26G or 36M treatment induced cell cycle arrest. The cell cycle was arrested in the SAS and OECM-1 cells at the S or G2/M phase after treatment with 26G and 36M for 48 h. The 26G induced S phase arrest and 36M induced S and G2/M phase arrest in SAS cells (Figure 2A). The S and G2/M phase arrest was induced by 26G, and 36M induced S phase arrest in OECM-1 cells ( Figure 2B). Western blotting was performed to analyze the relative protein expression during the cell cycle. The results showed that both SAS and OECM-1 cells treated with 26G or 36M at 100 µM significantly decreased the protein expression of CDK1, CDK2, cyclin D1, and cyclin B1 compared with the control group. Cyclin A2 expression was inhibited by 26G and 36M in SAS cells but not in OECM-1 cells. P21 was upregulated in SAS cells but downregulated in OECM-1 cells ( Figure 2C,D). The occurrence of cell cycle-related protein downregulation supports the result of 100 µM 26G and 36M inducing S and G2/M phase arrest in SAS and OECM-1 cells. However, 25 µM 26G treatment enhanced cyclin D1 and cyclin B1 expression ( Figure 2C) and the cell viability was not enhanced ( Figure 1A), which may be related to the antioxidant capacity of the cells [18].

The 26G and 36M Treatments Increased G1 Phase and S Phase Arrest
Analyzing the effects of 26G and 36M on the cell cycle is important for understanding their anticancer mechanisms. PI staining and flow cytometry were performed to analyze whether 26G or 36M treatment induced cell cycle arrest. The cell cycle was arrested in the SAS and OECM-1 cells at the S or G2/M phase after treatment with 26G and 36M for 48 h. The 26G induced S phase arrest and 36M induced S and G2/M phase arrest in SAS cells (Figure 2A). The S and G2/M phase arrest was induced by 26G, and 36M induced S phase arrest in OECM-1 cells ( Figure 2B). Western blotting was performed to analyze the relative protein expression during the cell cycle. The results showed that both SAS and OECM-1 cells treated with 26G or 36M at 100 μM significantly decreased the protein expression of CDK1, CDK2, cyclin D1, and cyclin B1 compared with the control group. Cyclin A2 expression was inhibited by 26G and 36M in SAS cells but not in OECM-1 cells. P21 was upregulated in SAS cells but downregulated in OECM-1 cells ( Figure 2C

After 26G and 36M Treatment, ROS Increased at 24 h and then Decreased at 48 h
CAPE is an antioxidant. However, whether or not 26G and 36M have antioxidant activities remains unclear. Total ROS stress was measured using the carboxy-H2DCFDA detection method. The ROS levels increased under the 25 µM and 50 µM 26G or 36M treatment, and decreased under the 100 µM 26G or 36M treatment for 24 h in SAS cells. However, the ROS levels decreased under 25 µM to 100 µM 26G or 36M treatment for 48 h in SAS cells ( Figure 3A). In OECM-1 cells, the ROS levels increased under the 25-100 µM 26G or 36M treatment for 24 h but decreased under 100 µM 26G or 50 µM, and 100 µM 36M treatment for 48 h ( Figure 3B). Overall, treatment with 26G below 50 µM or 36M below 100 µM for 24 h induced ROS generation in SAS and OECM-1 cells, respectively. SAS and OECM-1 cells showed similar trends in ROS production after 26G and 36M treatment, but SAS was more sensitive than OECM-1 was. The antioxidant activity of 36M was better than that of CAPE (26G) in both SAS and OECM-1 cells. Next, we analyzed the expression levels of antioxidant-associated proteins in both cell lines after treatment with 26G and 36M for 48 h. Nrf-2, CAT, HO-1, and superoxide dismutase type 1 (SOD-1) levels showed a decreasing trend in SAS cells treated with 26G and 36M ( Figure 3C, left panel). Treatment with 26G significantly reduced CAT expression levels and treatment with 36M reduced Nrf-2, CAT, and HO-1 expression levels in SAS cells ( Figure 3C, histograms). The Nrf-2, CAT, and NQO-1 also show a weakening trend in OECM-1 cells treated with 26G and 36M ( Figure 3D, left panel). The 26G treatment significantly reduced Nrf-2 and CAT expression, and the 36M treatment reduced Nrf-2, CAT, and NQO-1 in OECM-1 cells ( Figure 3D, histograms). Although antioxidant proteins were inhibited by the 26G and 36M treatments for 48 h in SAS and OECM-1 cells, total ROS increased at 24 h and then decreased at 48 h. These data show that OSCC cells were not protected by 26G and 36M antioxidant mechanisms. It has been speculated that 26G and 36M neutralize free radicals by accepting or donating electrons (s) to eliminate the unpaired condition of the radical [19]. A previous study suggested that patients with low ROS levels may be more sensitive to anticancer therapy [20]. Therefore, 26G or 36M may improve the efficacy of chemotherapy, but this needs to be confirmed in in vivo models.  Cell cycle distribution is shown in the bar graph. The vertical axis represents the percentage of cells in the G2/M, S, and G1 phases of the cell cycle, and the horizontal axis represents the concentration of 26G and 36M used. (C,D), the CDK1, cyclin A2, CDK2, cyclin D1, cyclin B1, and P21 expression determined by Western blotting analysis. GAPDH was used as the internal control. The cell cyclerelated proteins were quantified, and the results are presented in the right histogram. Data are expressed as mean ± SD; * p < 0.05, and ** p < 0.01 compared to the vehicle control group. SD, standard deviation.

Treatment with 26G and 36M
Induced Autophagy Via mTOR-ULK1-P62-LC3 Signaling Caused by ROS Generation ROS control and determine the type of cell death including autophagy, apoptosis, ferroptosis, and necroptosis [21]. CAPE induces autophagy in human SH-SY5Y neuroblastoma cells [22], breast cancer MDA-MB-231 cells [23], and colorectal adenocarcinoma WiDr cells [24]. In the OSCC cell lines, SAS and OECM-1, treatment with 26G and 36M increased the levels of phosphorylated ULK-1 and LC3-II proteins in a dose-dependent manner. However, the phosphorylated mTOR and p62 remarkably decreased after the 25-100 µM of 26G and 36M treatment in SAS ( Figure 4A) and OECM-1 ( Figure 4B) cells. Vitamin E (VE) is a common antioxidant. Pretreatment with vitamin E reduced the 26G and 36Mstimulated total ROS signals ( Figure 4C). Briefly, 26G and 36M down-regulated protein expressions; LC-3 and P62 were reversed by vitamin E pretreatment in SAS ( Figure 4D) and OECM-1 cells ( Figure 4E). These results suggest that 26G and 36M trigger the activation of autophagy due to increased cellular oxidative stress, which leads to oral cancer cell death.

Treatment with 36M Decreased Malignant Ability and Angiogenesis of Cancer Cells
26G and 36M induce cytotoxicity, cell cycle S or G2/M phase arrest, and autophagy in OSCC cells. Both SAS and OECM-1 cells were more sensitive to 36M than to 26G; therefore, the anti-OSCC potential of 36M requires further investigation. The SAS cells treated with 100 µM 36M, and the OECM-1 cells treated with 50 µM to 100 µM significantly reduced migration ability as compared with the corresponding control group after 4 or 8 h ( Figure 5A). Furthermore, the colony formation in the SAS and OECM-1 cells was significant inhibited following the treatment with doses of 25 µM to 100 µM of 36M ( Figure 5B). The HIF1-α expression has been well demonstrated it played a vital role in cancer progression, angiogenesis, and metastasis [19,20]. The anti-angiogenic abilities of 26G and 36 M were compared in SVEC 4-10 cells in vitro. After 100 µM 26G and 36M treatment, the microtube formation ability of SVEC 4-10 was inhibited. Partial microtube formation was still observed in the 26G treatment group, but was completely absent in the 36M treatment group ( Figure 5C). HIF1-α mRNA and protein expression in SVEC4-10 cells were also analyzed. The SVEC4-10 cells treated with 50 µM and 100 µM 26G or 36M for 24 h, the 26G suppressed HIF1-α protein only, and 36M suppressed both HIF1-α mRNA and protein expression levels ( Figure 5D,E).   -1 (B). Left panels, 24 h; right panels, 48 h. After 26G or 36M treatment for 48 h, the protein expressions of Nrf-2, CAT, HO-1, SOD-1, and NQO-1 in SAS (C) and OECM-1 (D) were analyzedvia Western blotting. The protein expression level quantification of each targeted protein under each treatment is shown in the histograms on the right. GAPDH was used as a loading control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the vehicle control group.

Treatment with 26G and 36M Induced Autophagy Via mTOR-ULK1-P62-LC3 Signaling Caused by ROS Generation
ROS control and determine the type of cell death including autophagy, apoptosis, ferroptosis, and necroptosis [21]. CAPE induces autophagy in human SH-SY5Y neuroblastoma cells [22], breast cancer MDA-MB-231 cells [23], and colorectal adenocarcinoma WiDr cells [24]. In the OSCC cell lines, SAS and OECM-1, treatment with 26G and 36M increased the levels of phosphorylated ULK-1 and LC3-II proteins in a dose-dependent manner. However, the phosphorylated mTOR and p62 remarkably decreased after the 25-100 μM of 26G and 36M treatment in SAS ( Figure 4A) and OECM-1 ( Figure 4B) cells. Vitamin E (VE) is a common antioxidant. Pretreatment with vitamin E reduced the 26G and 36M-stimulated total ROS signals ( Figure 4C). Briefly, 26G and 36M down-regulated protein expressions; LC-3 and P62 were reversed by vitamin E pretreatment in SAS (Figure 4D) and OECM-1 cells ( Figure 4E). These results suggest that 26G and 36M trigger the activation of autophagy due to increased cellular oxidative stress, which leads to oral cancer cell death.  . The 26G and 36M treatment for 24 h activated mTOR-ULK1-P62-LC signaling by ROS production in OSCC cell lines. The OSCC cells were treated with 26G or 36M for 24 h. Subsequently, the autophagy-relative protein expressions of p-mTOR, p-ULK-1, p62, and LC-3 I/II in SAS (A) and OECM-1 (B) were determined by western blot. The protein expression level quantification under each treatment is shown in the right histograms. The SAS or OECM-1 cells were pretreatment with vitamin E (20 µM) for 2 h, and the 26G or 36M (100 µM) was then added for another 24 h (C-E). (C) The intracellular ROS level was analyzed by the DCFDA approach. Fluorescence images were recorded under 100X magnification using a fluorescence microscope. Up panel, SAS; low panel, OECM-1. The protein expressions of LC3 in SAS (D) and OECM-1 (E) were determined by western blot, and the protein expression level quantification under each treatment is shown in the right histograms. The GAPDH was used as a loading control. * p < 0.05, ** p < 0.01 compared to the vehicle control group. + p < 0.05, ++ p < 0.01 compared to the 26G or 36M group alone.

Discussion
In the present study, we demonstrated that 26G and 36M could decrease malignancy owing to their ability to induce oral cancer cell death through mTOR-ULK1-P62-LC3 signaling. CAPE has been used in the treatment of patients with oral cancer and has increased their survival rate in 5 years [25]. The IC50 of CAPE were 129.7 ± 4.2 μM and 159.2 ± 7.2 μM in SAS and OECM-1, respectively [26]. Compare to our result, the IC50 of CAPE (129.7 ± 4.2 μM) was closed to 26G (54.0 μM) and 36M (161.3 μM) in SAS cells. However, the cytotoxicity will be affected by cell density, treatment time, serum percentage in the medium, and analysis methods, etc. Herein, the lower level of caffeamide derivative 36M treatment for 72 h presented a more potent ability to induce cell toxicity in cancer cells than the corresponding dose level of the CAPE derivative 26G (Figure 1A,B). This study indicates that 36M plays critical roles in preventing type 2 diabetes and oral microbes because of its broad resistance to acid, alkali, and high-temperature conditions [27,28]. Furthermore, 36M showed more potent cell cytotoxicity than other CAPE derivatives [28]. The overall results and mechanisms of 26G and 36M suppression in OSCC cells are shown in Figure 5F. 26G and 36M early induction of ROS production, but late induction decreased ROS production in OSCC cells. This induced ROS promotes the expression of NRF-2 and activates intracellular antioxidant proteins (CAT, HO-1, SOD-1, and NQO-1) to reduce ROS in a positive regulatory manner. Simultaneously, ROS can cause cytotoxicity and cell-cycle arrest. Cell cycle-related proteins, including p21, cyclin D1, cyclin B1, and CDK2, were inhibited. In addition, ROS induced autophagy signaling, such as p-mTOR and p62 downregulation and p-ULK-1 and LC3 I/II upregulation. 26G and 36M induced changes in p62 and LC3 I/II, which were reversed by vitamin E pre-treatment. Finally, ROS activates HIF-1α expression. Treatment with 26G and 36M inhibited HIF-1α expression and interfered with tumor angiogenesis. Based on these results, we concluded that 26G and 36M are bioactive substances with anti-OSCC potential.

Discussion
In the present study, we demonstrated that 26G and 36M could decrease malignancy owing to their ability to induce oral cancer cell death through mTOR-ULK1-P62-LC3 signaling. CAPE has been used in the treatment of patients with oral cancer and has increased their survival rate in 5 years [25]. The IC50 of CAPE were 129.7 ± 4.2 µM and 159.2 ± 7.2 µM in SAS and OECM-1, respectively [26]. Compare to our result, the IC50 of CAPE (129.7 ± 4.2 µM) was closed to 26G (54.0 µM) and 36M (161.3 µM) in SAS cells. However, the cytotoxicity will be affected by cell density, treatment time, serum percentage in the medium, and analysis methods, etc. Herein, the lower level of caffeamide derivative 36M treatment for 72 h presented a more potent ability to induce cell toxicity in cancer cells than the corresponding dose level of the CAPE derivative 26G (Figure 1A,B). This study indicates that 36M plays critical roles in preventing type 2 diabetes and oral microbes because of its broad resistance to acid, alkali, and high-temperature conditions [27,28]. Furthermore, 36M showed more potent cell cytotoxicity than other CAPE derivatives [28]. The chemical structure revealed that 26G was more hydrophilic than 36M because the polarity of the aromatic group in 26G was stronger than that of the 36M alkyl group ( Table 1). The solubility and diffusion rates of 26G were low at 36M, which could explain why the anticancer activity of 36M was more potent than that of 26G [28].
Based on the histograms shown on the right side of Figure 4C,D, it is evident that treatment with 25-100 µM of 26G and 36M led to a decrease in the expression of CDK1, CDK2, and cyclin B1 in OECM-1 and SAS cells. However, the treatment with 25 µM of 26G induced cyclin B1 expression in SAS cells but not in OECM-1 cells. The expression of cyclin A2 showed inconsistency after treatment in both cell lines. This variability may be attributed to the different p53 statuses in OECM-1 and SAS cells. The tumor suppressor proteins p53 and p21 play a crucial role in inhibiting cell cycle progression. Mutations in the p53 gene have been detected in over 70% of human cancers. In particular, OECM-1 cells exhibited a point mutation at codon 173 (GTG to TTG), while SAS cells had a point mutation at codon 305 (GAG to a TAG stop codon) in their p53 genes. The molecular weights of the p53 protein were 53 kD in OECM-1 and 40 kD in SAS, indicating abnormal p53 functions in both cell lines. However, it is important to note that mutated p53 is involved in various cellular processes such as DNA repair, cell cycle regulation, programmed cell death, and senescence. Further investigation is required to understand the function of mutated p53 in OECM-1 and SAS cells [29].
LC3 is a well-known autophagy marker. When LC3-I is hydrolyzed by autophagyrelated 4 (ATG4) from LC3-I, it is converted into LC3-II, termed the autophagosome, which fuses with the lysosome to form an autolysosome, leading to the degradation of cytoplasmic materials [30,31]. A previous study demonstrated that CAPE enhanced the autophagic response to the induction of cancer cell death by AMP-activated protein kinase (AMPK) inactivation in C6 glioma cells [32]. Another study showed that CAPE increased intracellular ROS levels by altering cellular oxidative and endoplasmic reticulum (ER) stress responses, triggering autophagy in SH-SY5Y cells [22]. In the present study, we also found that treatment with 26G and 36M led to the activation of autophagic signaling in SAS and OECM-1 cells (Figure 4). Moreover, phosphorylated p62 has been reported to play an important role in the autophagy response owing to its association with autophagylysosome degradation. During autophagy, p62 cannot be phosphorylated by optineurin (OPTN) or TANK-binding kinase 1 (TBK1), which prevents cytoplasmic materials from being degraded and deposited within the cytosol [33]. Herein, non-phosphorylated p62 protein was reduced by treatment with 26G or 36M for 24 h in oral cancer cells, remarkably increasing LC3-II expression (Figure 4), which suggests that the autolysosome was formed for cytoplasmic material degradation by 26G and 36M stimulation in a relatively late stage of autophagy.
In colon carcinoma, the inhibition of catalase to enhance intracellular levels of free radicals leads to cell death, which might provide a new concept to the clinical therapy of drugs [34]. In this study, cellular ROS levels were altered by 26G and 36M treatments in OSCC cells ( Figure 3A,B). However, the oxidative-stress relative protein Nrf-2 and catalase were significantly decreased by 26G and 36M treatments ( Figure 3C,D). A previous study showed that Nrf-2 is a nuclear transcription factor that increases the transcription and translation of the catalase protein in the cell [35]. In the antioxidant system, SOD is the primary defense enzyme for the chelation of superoxide (O2 •-) to H 2 O 2 , and then, H 2 O 2 is converted to H 2 O and O 2 by catalase [36]. This defense system inhibits normal cells or leads to cancer cell death [36,37]. Hence, treatment with 26G and 36M caused unbalanced cellular oxidative stress, inducing cell death, which was not chelated by catalase upon correlation with cellular ROS (Figures 1 and 3). Additionally, it is well-known that free radical generation causes lipid peroxidation and DNA damage or mitochondrial dysfunction to induce cell death in a dysfunctional antioxidant defense system [38]. Therefore, our results suggest that oral cancer cell death is linked to cellular free radical production and the downregulation of catalase protein expression under 26G and 36M treatments.
The accumulation of ROS in oral cancer cells was measured 24 h after 26G and 36M treatments. We found that ROS levels decreased due to cell death at 48 h (Figures 1 and 3A,B). At this time, 26G and 36M treatments inhibited the expression of the transcriptional activator NRF2 from suppressing downstream anti-ROS gene transcription. CAT, HO-1, SOD-1, and NQO-1 protein expression decreased in a dose-dependent manner ( Figure 3C,D). Therefore, 26G and 36M did not exert antioxidant activity in oral cancer cells but induced ROS generation in a short time. Increased cellular ROS levels activated apoptosis and autophagy in the tongue squamous cell carcinoma cells [39]. In the present study, the mTOR-ULK1-LC3 signaling pathway was upregulated by the 26G and 36M treatments at 100 µM for 24 h (Figure 4A,B). Vitamin E is an antioxidant that reduces the production of ROS. We used vitamin E to block intracellular ROS generation and decrease LC3 expression in SAS and OECM-1 cells under 26G and 36M treatments ( Figure 4C-E). Our results indicated that 36M increases autophagy by mediating mTOR-ULK1-P62-LC3 signaling in OSCC cells.
Approximately 90% of cancer-related deaths occur in patients with cancer cell metastasis [40]. Metastasis accelerates the mortality rate of patients with oral cancer [41]. In other words, preventing metastatic colonization could improve the survival rate of oral cancer patients, which may benefit clinical treatment [42].In fact, 26G and 36M inhibited malignant phenotypes, including cell migration, colony formation, and angiogenesis ( Figure 5).
Longitudinal clinical investigations showed that combined supplementation of herbal medicines with vitamin E efficiency reduced the mortality rate in lung cancer patients in 5 years [43,44]. Therefore, supplements containing bioactive herbal compounds may provide more efficient therapies for patients with cancer. Compounds 26G and 36M show potential as effective treatments for oral cancer by increasing oral cancer cell toxicity and preventing metastasis, which may increase the survival rate of patients with oral cancer after treatment.

Compound Purification
CAPE and caffeamide derivatives (26G, 36C, 36H, 36 K, and 36M) were synthesized and provided by Dr. Yueh-Hsiung Kuo, and the extraction processes were as reported previously [27,45]. Briefly, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate was dissolved in dichloromethane and added to a mixture of caffeic acid, phenethyl alcohol, corresponding amines, and triethylamine in dimethylformamide. The reaction mixture was evaporated under a vacuum and partitioned between ethyl acetate (EtOAc) and H 2 O. The EtOAc layer was washed with 3N HCl and 10% NaHCO 3 and concentrated under a vacuum. All products were >95% purified via silica gel column chromatography (70-230 and 230-400 mesh, Merck, Burlington, MA, USA). All caffeamide derivatives were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 • C.

Cell Culture
The two OSCC cell lines, SAS and OECM-1, were kindly provided by Professor Hsi-Feng Tu from the National Yang-Ming Chiao Tung University. SAS cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. OECM-1 cells were cultured in a RPMI-1640 medium containing 10% FBS and 1% penicillin and streptomycin solution. The mouse vascular endothelial cell line SVEC4-10 was purchased from the Bioresource Collection and Research Center (BCRC: 60220, Hsinchu, Taiwan). The SVEC4-10 cells were cultured in DMEM containing 10% FBS, 4 mM glutamine, and 1% penicillin and a streptomycin mixture solution. All the cell lines were cultured at 37 • C in a 5% CO 2 atmosphere, as described previously [46,47]. When the cells reached approximately 90% confluence, they were treated with 26G or caffeamide derivatives at different times and doses depending on the experimental design. DMSO was used as the vehicle control.

Cell cycle Arrest Determination
SAS and OECM-1 cells (5 × 10 5 cells) were cultured in 6 cm cell culture dishes for 20-24 h. The cells were then treated with the vehicle or various concentrations of 26G or 36M for 48 h. Next, the cells were fixed with 70% ethanol at −20 • C overnight and stained with propidium iodide/Triton X-100 mixtures at room temperature (~25 • C) for 30 min. The cell cycle distribution was determined using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) [29].

Intracellular ROS Level
SAS and OECM-1 cells (5 × 10 5 cells) were cultured in 6 cm cell culture dishes for 20-24 h. The cells were then treated with vehicle or various concentrations of 26G or 36M for 48 h. Subsequently, the cells were incubated with 20 µM 5-(and 6)-Carboxy-2 ,7 -dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) in a serum-free medium at 37 • C for 45 min in the dark. Finally, cellular ROS levels were detected using a flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence images were captured using a fluorescence microscope (Eclipse TS100, Nikon, Minato, Tokyo, Japan).

Western Blot
Western blotting was performed using 50 µg proteins, as described previously [50]. Cell lysates were electrophoresed on a 10% polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% nonfat milk for 1 h and then incubated with primary antibodies at 4 • C overnight (Supplementary Table S1). After washing, the membranes were incubated with a secondary antibody (Abcam, Cambridge, UK) for 1 h. GAPDH was used as the internal control. Chemiluminescence imaging was performed using the SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA). Densitometric estimates were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The cells were treated with vehicle or various concentrations of 26G or 36M, and observed at different time points. The wounded area was observed and photographed using a microscope and quantified by wound width using ImageJ software (National Institutes of Health, Bethesda, MD, USA) [29].

Colony Formation
For the colony formation assay, SAS 2 × 10 3 cells/3 mL and OECM-1 5 × 10 2 cells/ 3 mL cells were incubated in each well of 6-well tissue culture plates and cultured at 37 • C under 5% CO 2 for 20-24 h. Cells were treated with the vehicle or various concentrations of 26G or 36M in a medium supplemented with 10% FBS for 24 h. The old medium was replaced with a fresh culture medium, and cells were then cultured for another 7 days to allow single cells to form colonies. At the end of the experiment, the colonies were fixed in methanol and stained with 0.01% crystal violet for 20 min at room temperature. Subsequently, the colonies were randomly photographed in 5 fields per well. Colonies containing > 50 cells were counted in each field. The number of colonies formed in each well was calculated [29].

Tube Formation Assay
Matrigel (50 µL; BD Biosciences; 354234) was added into 96-well plates and incubated at 37 • C for 1 h for gel formation. Next, 1 × 10 5 SVEC4-10 cells were incubated on colloidal Matrigel and treated with the vehicle or various concentrations of 26G and 36M. Random photographs of tube formation were taken for each well for 2-4 h after 26G and 36M stimulation [47].

Statistical Analysis
All experiments were performed at least three times independently. All experimental results were analyzed using Student's t-test using SPSS software (IBM, Armonk, NY, USA), where a p-value of < 0.05 represents a significant difference.

Conclusions
We concluded that ROS played a dominant role in inducing cell death under 26G and 36M treatments by regulating the mTOR-ULK1-P62-LC3 signaling pathway. Thus, 26G and 36M might be used to treat patients with oral cancer.